Thyroid peroxidase sequence has been previously accorded GenBank Accession No. J02969.
The present invention relates generally to thyroid peroxidase and, more specifically, to autoantibody epitopic regions of thyroid peroxidase and diagnostic and therapeutic applications thereof.
Thyroid peroxidase (TPO) is the major thyroid-specific membrane autoantigen implicated in Hashimoto""s thyroiditis, an autoimmune disease characterized by intense lymphocytic thyroiditis resulting in the destruction of the thyroid. Weetman, A. P. et al, Endocrinol. Rev. 5:309-355 (1984). TPO""s role in autoimmune disease and the precise localization of autoantibody binding has, however, been a subject of much debate. For example, there has been controversy as to whether autoantibodies inhibit thyroid peroxidase activity, thereby contributing to thyroid dysfunction in autoimmune thyroid disease. See Yokoyama, N. et al., J. Clin. Endocrinol. Metab. 68:766-773 (1989): Saller, B. et al., J. Clin. Endocrinol. Metab. 72:188-195 (1991). Reports on variations in the structure of TPO have suggested alternative mRNA splicing as a source of antigenicity. Kimura, S. et al., PNAS (USA) 84:555-5559 (1987); Nagayama, Y. et al., J. Clin. Endocrinol. Metab. 71:384-390 (1990); Zanelli, E. et al., Biochem. Biophys. Res. Commun. 170:735-741 (1990). The appearance of these alternative transcripts in normal thyroid glands has, however, brought the relationship of alternative splicing and autoimmunity into question. Elisei, R. et al., J. Clin. Endocrinol. Metab., 72:700-702 (1991). It also now appears that carbohydrate structures are not involved in TPO antigenicity. Foti, D. et al., Endocrinol. 126:2983-2988 (1990).
Recent reports on the number and type of autoantibody epitopes found in human TPO have been conflicting. One group has suggested that TPO autoantibodies bind only to conformation epitopes and that no localized epitopes exist. Finke, R. et al., J. Clin. Endocrinol. Metab. 71:53-59 (1990). Given that this group demonstrated binding to recombinant TPO produced in CHO cells, but was unable to show binding to whole recombinant TPO in the bacterial expression system employed, the failure to identify localized epitopes could have resulted from a problem with the expression of intact TPO in bacteria. In contrast, another group has reported the identification of localized autoantibody of TPO epitopes using in vitro translated TPO cDNA clones. Ubert, F. et al., EMBO J. 6:4193-4196 (1987); Ludgate, M. et al., J. Clin. Endocrinol. Metab. 68:1091-1096 (1989). Using sera from Hashimoto""s disease patients, localized autoantibody binding was identified between amino acids 590 and 675 of the TPO coding sequence. Id. While initially identifying only a single antibody binding site in this region, more recent publications of this group indicated the presence of multiple antibody binding sites in the carboxyl half of the TPO molecule. Elisei, R. et al., Autoimmunity 8:65-70 (1990); Libert, F. et al., J. Clin. Endocrinol. Metab. 73:857-560 (1991). Several other studies using trypsin digests of purified native TPO have also suggested the presence of multiple autoantibody epitopes, including some which appear to be conformational and require disulfide bonds. Nakajima, Y. et al., Mol. Cell Endocrinol 53:15-23 (1987); Yokoyama, N. et al., J. Clin. Endocrinol. Metab. 70:758-765 (1990). However, there has been no consensus on the exact number and type of epitopes present in TPO.
The identification of autoantibody thyroid-specific epitopes is important to the understanding and diagnosis of autoimmune thyroid disease and in developing effective immunotherapeutic strategies against thyroid disease and cancer. The identification of specific localized TPO epitopic regions would provide a powerful diagnostic tool for autoimmune diseases such as Hashimoto""s to distinguish it from other thyroid conditions. The identification and ability to produce sequences encompassing specific TPO autoantibody epitopes would also be instrumental in developing immunotherapeutic strategies against autoimmune thyroid disease and thyroid cancer.
The development of new therapeutic approaches to treating thyroid cancer is of particular importance. Thyroid cancer is diagnosed in 10,000 individuals in the United States each year. Mazzaferri, E. L. xe2x80x9cThyroid Cancer,xe2x80x9d Ch. 43:319-331 (Becker, K. L. ed.) Principles and Practice of Endocrinology and Metabolism, J. B. Lippencott (Philadelphia Pa. 1990); Cooper, D. S. et al., Encrinol. Metab. Clin. North Am. 19(3):577-591 (1990). While modern techniques have led to early diagnosis and treatment, approximately 10% of thyroid cancer patients develop terminal metastatic disease. Robbins, J. et al., Ann. Intern. Med. 115(2):133-147 (1991). Thyroid cancer presents an important public health problem in that it most commonly occurs in otherwise healthy and productive individuals in the third and fourth decades of life. Mazzaferri, E. L. xe2x80x9cThyroid Cancer,xe2x80x9d Ch. 43:319-331 (Becker, K. L. ed.) Principles and Practice of Endocrinology and Metabolism, J. B. Lippencott (Philadelphia Pa. 1990). It is also an extremely important health problem for women, as it occurs three times as often in women than in men. Kramer, J. B. et al., Adv. Surg. 22:195-224 (1989). The disease can also be seen in children, especially after radiation exposure. Barnes, N. D., Horm. Res. 30:84-89 (1989). While less common in the elderly, thyroid cancer has a much worse prognosis in this population. Mazzaferri, xe2x80x9cThyroid Cancer,xe2x80x9d Ch. 43:319-313 (Becker K. L., ed.) Principles and Practice of Endocrinology and Metabolism (J. B. Lippencott, Philadelphia Pa. 1990); Cooper, D. S. et al., Endocrinol. Metab. Clin. North Am. 19(3):577-591 (1990).
Current therapeutic approaches for the two most prevalent types of thyroid cancer, papillary and follicular carcinoma, involve partial or complete thyroidectomy, often in conjunction with radioactive iodine therapy. However, a substantial number of patients show no response to conventional treatment For example, some thyroid tumors do not respond to radioactive iodine treatment because the carcinoma is not sufficiently differentiated to concentrate lethal quantities of radioactivity. Additionally, the side effects attending conventional chemotherapeutic and radiation therapy have a serious adverse impact on patient health and well-being.
More sensitive diagnostic and alternative therapeutic strategies against thyroid disease and cancer would thus be desirable. Such strategies can be provided with the identification of specific thyroid peroxidase epitopic regions.
Specific epitopic regions of thyroid peroxidase (TPO) (SEQ ID NO:2) a thyroid-specific cell membrane enzyme and the autoantibody target in autoimmune thyroid disease, have been identified. The epitopic regions reside from amino acid 456 to 933, with two distinct regions located from amino acid 517 to 630 and from amino acid 633 to 933, the former region having at least one distinct binding region therewithin located from amino acid 592 to 613 inclusive. The latter region also has at least two distinct binding regions therewithin, located from amino acid 633 to 768 and from amino acid 768 to 933. The specific epitopic regions provide a sensitive diagnostic reagent for autoimmune thyroid disease and, as thyroid-specific structures, can be used for immunotherapy of thyroid disease and cancer. Nucleic acid sequences coding for TPO epitopic regions, and sequences complementary thereto, are also contemplated as being within the scope of the present invention.
Since TPO is expressed in most thyroid cancers, patients can be immunized with TPO epitope contained within these regions to induce or augment the immune response to TPO, specifically targeting the TPO-bearing cancer cells. Adoptive immunotherapy involving the harvesting of immune cells from the thyroid, their in vitro stimulation by TPO epitope and reimplantation into the patient is also now feasible. Monoclonal antibodies specific for TPO epitope/epitopic region can also be administered for passive immunotherapy.
Autoimmune thyroid disease, such as Hashimoto""s thyroiditis, can also be treated by blocking or competing with autoantibody binding to native TPO in the thyroid. More sensitive assays utilizing TPO epitopic binding to screen for the presence of autoantibody are also useful in diagnosing patients with Hashimoto""s disease and in distinguishing Hashimoto""s from other thyroid disorders.
Other features and advantages of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings.